Lysyl-tRNA Synthetase from Bacillus stearothermophilus. Formation and Isolation of an Enzyme•Lysyladenylate Complex and Its Analogue

抄録

The formation of an enzyme. lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase [L-lysine:tRNA^Lys ligase (AMP-forming); EC 6.1. 1.6] from Bacillus stearothermophilus. The apparent dissociation equilibrium constants of the enzyme with L-lysine and ATP in the process of the complex formation were estimated to be 50.9 and 15.5 μM, respectively, at pH 8.0, 30°C, by fluorometric measurement. The isolated enzyme•lysyladenylate complex was relatively stable with a rate constant of decomposition of 1.7×10^-5 s^-1 at pH 8.5 and 0°C The rate constant of transfer of L-lysine from the complex to Escherichia coli tRNA was 1.2×10^-2 s^-1 at pH 8.5 and 0°C. The effects of replacing L-lysine by several analogues on the complex formation were examined. L-Lysine hydroxamate, a strong inhibitor of the L-lysine dependent ATP-PP₁ exchange reaction, produced a stable complex with the enzyme and ATP, enzyme•lysinehydroxamate-AMP probably being formed. The binding stoichiometry of the assumed L-lysinehydroxamate-AMP per mol of the dimer enzyme was 1:1.